The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. This study concerns the coordination of the activities of these two kinases in vivo. INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. This study shows that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. The results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation (Carmena, 2012).

Coordination of Polo and Aurora B activity at kinetochores is critical in early mitosis, as the two kinases play potentially antagonistic but complementary roles in regulating kinetochore-microtubule interactions. Aurora B is essential for the correction of aberrant attachments, and indeed, tethering Aurora B too close to kinetochores interferes with the formation of stable attachments. In contrast, Plk1 activity is required for initial stabilisation of microtubule attachments to kinetochores. It is suggested that interactions with INCENP may provide a mechanism to coordinate the activities of these two essential kinases during early mitosis (Carmena, 2012).

Recent studies suggest that Plk1 is activated at centrosomes when its T-loop (T210) is phosphorylated by Aurora A kinase-Bora, and that this promotes the G2/M transition upstream of Cdk1, although Polo activity is not required for mitotic entry. How Plk1 is activated at kinetochores remained an important unsolved question. The present results show that Aurora B and INCENP, which are concentrated at inner centromeres, function there to activate Polo by phosphorylating its T-loop (Carmena, 2012).

Plk1 recruitment to centromeres in late G2 has been variously proposed to be mediated by Bub1, INCENP, and BubR1. Another report implicated the self-primed interaction of Plk1 with PBIP1/CENP-U. This could potentially explain why Plk1 activity is reportedly required for its localisation to kinetochores in human cells (Carmena, 2012).

The current RNAi studies confirmed that Plk1 is partially dependent on the CPC for its centromeric localization in human cells. However, this appears not to be the case in Drosophila, where Polo is present at centromeres before NEB, at a time when INCENP is not yet concentrated at inner centromeres and before PoloT182ph, the active form of the kinase, is detected there. Indeed, no significant decrease was observed in kinetochore-associated Polo levels after INCENP RNAi in Drosophila cells (Carmena, 2012).

Although Polo targeting to kinetochores is independent of the CPC in Drosophila, its activation there does require the CPC with active Aurora B. The data suggest that INCENP binding to Polo facilitates its subsequent activation by Aurora B kinase. Indeed, INCENP and Polo interact physically in vitro and co-immunoprecipitate in mitotic cell extracts. Although most centromeric Polo kinase is concentrated in the outer kinetochore in prophase and prometaphase, active Polo (PoloT182ph) is also found in inner centromeres, where it overlaps with INCENP as confirmed by a proximity ligation assay (PLA)(Carmena, 2012).

A range of evidence presented in this study suggests that Aurora B is the upstream kinase responsible for Polo kinase activation at centromeres. Firstly, Aurora B phosphorylates Polo at Thr182 in vitro. Secondly, RNAi depletion of INCENP or Aurora B, but not Aurora A, reduces levels of active PoloT182ph at kinetochores. Thirdly, tissue culture cells and third larval instar neuroblasts treated with a specific inhibitor of Drosophila Aurora B kinase show decreased levels of PoloT182ph at kinetochores. In all of the preceding experiments, PoloT182ph levels are affected at kinetochores but not at centrosomes, where Polo is presumably activated by Aurora A. Importantly, this involvement of Aurora B in Polo activation at centromeres discovered in Drosophila is conserved for Plk1 in human cells (Carmena, 2012).

The current results suggest a model for interactions between Polo kinase and the CPC at centromeres (see Model for the interactions between the CPC and Polo kinase at the centromere/kinetochore). In Drosophila cells, Polo targets to centromeres before the CPC is recruited by Survivin binding to histone H3T3ph (Yamagishi, 2010: see Schematic depiction of the pathways that regulate CPC targeting to centromeres). At inner centromeres of chromosomes whose kinetochores are not under tension, Polo now binds to INCENP. This promotes Polo kinase activation, as Aurora B phosphorylates PoloT182. It is suggested that interactions with INCENP link the two complementary kinase activities, thereby potentially creating a microtubule attachment/detachment cycle at kinetochores. Such a cycle would not be possible without a balancing phosphatase activity, and PP2A-B56 has recently been shown to oppose both Aurora B and Plk1 activities at kinetochores to promote stable attachments (Carmena, 2012).

At metaphase, when chromosomes are bioriented and under tension, the CPC and Polo kinase exhibit only a partial overlap. A weakening of the INCENP/Polo PLA signals in metaphase suggests that Polo may be released from INCENP after its activation—possibly moving to the outer kinetochore. During metaphase, the CPC localizes in the inner centromere, stretching between sister kinetochores, whereas Polo and PoloT182ph concentrate mainly at the kinetochores. This separation may be necessary to allow Polo-mediated stabilisation of kinetochore-microtubule attachments. The coordinated activities of both kinases at kinetochores and their tension-mediated separation might facilitate a dynamic equilibrium between attached and unattached kinetochores, selectively stabilizing proper chromosome attachments (Carmena, 2012).

In summary, the results reveal that INCENP and Aurora B are responsible for Polo kinase activation at centromeres but not at centrosomes during mitosis. These experiments support the hypothesis that INCENP acts as a scaffold integrating the cross-talk between these two important mitotic kinases (Carmena, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

borr is ubiquitously expressed in the early Drosophila
embryo, suggesting maternal expression, although it appears to be restricted to the VNC and brain during later embryonic stages (Hanson, 2005).

In order to observe the subcellular localisation of Borr, Drosophila DmD8 cells were transfected with a construct encoding GFP-tagged full-length Borr. As expected, GFP-Borr is associated with chromatin during prometaphase (Eggert, 2004), and is subsequently concentrated at the central spindle midbody and at the cell cortex in the cleavage furrow during telophase and cytokinesis. This pattern will be referred to as 'localisation to the mitotic spindle'. Significantly, GFP-Borr colocalises with both endogenous Aurora B and Incenp, in agreement with the results by Eggert (Eggert, 2004), who also observed co-localisation of Borr and Aurora B throughout mitosis. These results are consistent with Borr being a CPC component, like its vertebrate counterparts (Hanson, 2005).

Effects of Mutation or Deletion

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. Parallel chemical genetic and genome-wide RNA interference screens were performed in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, a small molecule was identified that inhibits the Aurora B kinase pathway. The protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which have been identified. Dissection of the Aurora B pathway, where a new gene and a specific small molecule inhibitor were found, should prove particularly beneficial. This study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways (Eggert, 2004).

Aurora B, INCENP, and Survivin form the chromosomal passenger complex, which also includes CSC-1 in C. elegans and Borealin/Dasra B in humans. Aurora B kinase plays a number of roles during mitosis, including phosphorylating Histone H3 on Ser-10 and detecting errors in chromosome attachment in mitosis, and performs an essential, but poorly understood, function in cytokinesis. Chromosomal passenger proteins localize to the inner centromere during mitosis and move to the interzonal microtubules, the cleavage furrow, and eventually the midbody during cytokinesis. Because the sequences that targeted CG4454 and aurora B both had 21-bp overlaps with other genes in the dsRNA collection that was screened, dsRNA targeting different areas of these two genes were performed, and no change in phenotype was oberved. Since RNAi depletion of the new gene discovered in the screen, CG4454, resulted in the same phenotype as depletion of aurora B and INCENP, it was hypothesized that CG4454 could be a new member of the chromosomal passenger complex. Green fluorescent protein (GFP) fusion proteins were constructed to both C- and N-termini of CG4454. CG4454-GFP exhibited the signature localization of a passenger protein and co-localized with Aurora B throughout mitosis and cytokinesis, suggesting that it might be complexed to Aurora B. RNAi depletion of CG4454 or aurora B resulted in an absence of phosphorylated Histone H3 on mitotic chromosomes, further supporting the participation of CG4454 in the chromosomal passenger complex. Although CG4454 amino acid sequence reveals a remote similarity with Borealin/Dasra B (Gassmann, 2004), it is unclear at this point whether CG4454 is its Drosophila homolog. Unlike CG4454, RNAi depletion of Borealin does not significantly reduce Histone H3 phosphorylation (Gassmann, 2004). It might not be possible to confirm whether CG4454 and Borealin are related until structural information becomes available. However, to prevent further confusion in naming conventions, CG4454 has been named Borealin-related (Eggert, 2004).

borr is an essential gene required for embryonic mitoses

E133 is a loss-of-function allele of CG4454, with a single base pair
deletion at position 290 in the first exon of its coding region. The resulting
frameshift introduces a stop codon immediately after this deletion into the
predicted protein, truncating it after serine 98. Zygotic homozygosity for the borr mutation results in late
embryonic lethality, but the mutant embryos lack overt morphological defects,
probably owing to rescue by maternal gene product (Hanson, 2005).

Given its high expression levels in the embryonic nervous system, this tissue was
scrutinised carefully, after staining embryos with Hoechst
dye. Indeed, by stage 12, cells in the VNC and brain were detected with
abnormally large nuclei. It is estimated that the volumes of the borr mutant VNC nuclei are on average ~3 times larger than those of wild-type VNC
nuclei. This implies
an increased DNA content (>2N) of the mutant cells, and suggests that
borr loss affects the divisions of VNC cells. Similarly oversized nuclei were detected in other tissues (in addition to severe
morphological defects such as failure of germ band retraction), after
injection of borr dsRNA into wild-type embryos, which potentially
also depletes maternal gene product. Thus, borr loss
appears to affect many, if not all, dividing cells in the embryo (Hanson, 2005).

To monitor the mitotic events that are affected in the borr mutant
embryos, these embryos were stained with an antibody against serine 10
phosphorylated histone H3 (P-H3), a histone modification specifically found in
mitotic cells that has been ascribed to Aurora B kinase activity in several
organisms, including Drosophila. Counting
the mitotic cells per hemi-neuromere in wild-type and borr mutant
embryos, it was found that these numbers are reduced significantly in the
mutants, to ~50% of the wild type at stage 12, and to ~20% at stage 14. These estimates suggest that, in mutant embryos, the overall number of cells per hemi-neuromere is also lower than normal (although it is technically difficult to obtain accurate counts of total cell numbers). Nevertheless, these counts suggest
that the fraction of mitotic cells (i.e., the mitotic index) in the VNC of
borr mutant embryos may be reduced compared with the wild type (Hanson, 2005).

To see whether the borr mutation affects a specific mitotic stage,
each P-H3-positive cell was classified as one of four different mitotic stages
(based on the shapes of their chromatin masses), and
the frequencies of these stages were determined as a percentage of the total of mitotic cells. This revealed that the percentages of prophase and prometaphase cells were higher in borr mutants compared with the wild type, whereas anaphases and telophases were underrepresented in the mutants. This profile shift
of the mitotic stages appears to be progressive during embryonic development,
and becomes more pronounced by stage 14 when telophases have become
exceedingly rare, maybe as a result of cumulative defects during consecutive abnormal cell divisions. This profile shift suggests that borr loss causes a severe attenuation, or block, prior to metaphase (Hanson, 2005).

Two further features were noticeable in the P-H3 staining patterns of the
borr mutant VNC cells. (1) Many of the rare anaphases detected at
stage 12 appeared abnormal, showing evidence of uneven segregation of
chromatin. (2) The P-H3 staining intensity was reduced markedly, which is particularly noticeable during metaphase, but also during telophase when P-H3 staining normally fades away. These observations are consistent with the profile shift of the mitotic stages in borr mutant embryos, and they underscore the notion that the first major defect during the mutant cell cycle occurs prior to metaphase. A similar prometaphase block has been reported for human Borealin (Gassmann, 2004) and for other CPC components (Adams, 2001; Giet, 2001) in Drosophila cells (Hanson, 2005).

To further study the function of borr during mitosis, dsRNA interference was used in Drosophila Kc167 tissue culture cells. Indeed,
72 hours after addition of borr-specific dsRNA, Kc167 cells displayed
a range of mitotic defects when compared with their controls. Most notably,
highly abnormal multipolar spindles were observed in mitotic cells, and interphase
cells often showed single large nuclei -- reminiscent of the VNC nuclei
in borr mutant embryos -- or became multi-nucleate. Some of these
cells appear to have up to eight distinct nuclei, in addition to DNA fragments
strewn around the cytoplasm. Similar phenotypes were observed in HeLa cells after
RNAi-mediated depletion of Borealin, and also after RNAi-mediated depletion of
CPC components in Drosophila cells (Adams, 2001; Eggert, 2004;
Gassmann, 2004; Giet, 2001; Sampath, 2004). They
support the notion that Borr is a functional ortholog of human Borealin.
Furthermore, the multi-nucleate cells and the multipolar spindles suggest that
Borr is required for faithful segregation of chromosomes during mitosis, and
that its loss can cause polyploidy and/or aneuploidy (for simplicity, this will be referred to as 'polyploidy') (Hanson, 2005).

One crucial role of the CPC during mitosis is to mediate the H3
phosphorylation of serine 10 (P-H3) by Aurora B, as has been demonstrated in
budding yeast, C. elegans and Drosophila (Adams, 2001; Giet, 2001;
Hsu, 2000). The numbers of P-H3-positive (dividing) cells are reduced in the VNC of borr mutant embryos. Furthermore, the P-H3 levels of individual borr mitotic nuclei are typically reduced compared with those of wild-type nuclei. Often, they exhibit blotchy P-H3 staining rather than the more 'structured' staining outlining condensed chromosomes as observed in the wild type. A similar loss of P-H3 staining has also been observed in borr RNAi-depleted Kc167 cells (Eggert, 2004). This reduction of the P-H3 levels in borr mutant cells is consistent with a loss of Aurora B kinase activity and, thus, with a disruption of CPC function (Hanson, 2005).

Despite the strong reduction of the P-H3 levels in mitotic VNC cells of
borr mutant embryos, these cells display only a slight
undercondensation of their chromatin, although the degree of
undercondensation is somewhat variable from cell to cell. These
results suggest that borr may not be essential for chromatin
condensation (Hanson, 2005).

To examine the effects of borr loss on actively dividing
epithelial cells, FRT-FLP-mediated recombination was used to
generate borr mutant clones in imaginal discs whose cells undergo
cell divisions throughout larval development. If borr mutant clones
are induced during early larval stages and examined in fully grown larval
discs, these clones are rare and are much smaller than the corresponding
wild-type twin spots, suggesting that a large fraction of the mutant cells die. Hoechst staining revealed that many of the surviving
borr mutant cells are large, with giant but well-formed nuclei that
appear healthy, and well integrated into the epithelial tissue (Hanson, 2005).

Imaginal discs bearing borr mutant clones were stained with
antibodies against Incenp and Aurora B, to assess the effect of borr
loss on these CPC components during mitosis. Wild-type cells in metaphase show
characteristic well-ordered mitotic spindles, with distinct staining of Aurora
B and Incenp at specific sites along condensed chromatin.
By contrast, borr mutant cells invariably show abnormal mitotic
spindles, including multipolar ones. Most of these mutant spindles do not show any
chromatin-associated Incenp or Aurora B staining, although
occasionally patches of Incenp staining can still be observed, but they do not
seem to be associated with any of the spindle components. These
staining patterns suggest that these CPC components fail to localise properly
to mitotic spindles in the absence of borr (and their levels may also
be reduced, though the low frequency of surviving borr mutant cells
does not allow assessment of this quantitatively). Therefore, as in mammalian
cells, the correct localisation of Incenp and Aurora B to mitotic spindles of
dividing imaginal disc cells depends on Borr. This is further evidence that
Borr is a CPC protein, and that it interacts functionally with other known CPC
components (Hanson, 2005).

Borr loss causes delayed apoptosis of imaginal disc cells

Early-induced borr mutant clones are rare,
and are much smaller than their twin spots. Indeed,
many twin spots do not appear to have mutant cells associated with them,
indicating that the mutant cells have all died. The frequency of surviving
borr mutant clones is increased if they are induced in a
Minute background, which provides the mutant cells with a
proliferative advantage. They can thus occupy a significant fraction of
imaginal disc territories in third instar larvae. All discs are equally affected, and they tend to be smaller than wild-type discs at an equivalent stage. Larvae with these clones do not survive pupariation (Hanson, 2005).

Closer examination of the borr mutant cells revealed essentially
two distinct phenotypes: large cells with giant well-formed nuclei, and cells that appear to be undergoing apoptosis. The clearest
examples of the latter show compacted almost perfectly spherical nuclei that
are found at the basal-most level of the disc epithelium, well separated from
the healthy nuclei of the wing pouch. borr mutant cells were observed that
may be at an earlier step in the apoptotic process: their nuclei are less
compacted, and they are just beginning to drop basally within the epithelium. Antibody staining against active caspase 3 confirmed that the borr mutant
cells with compacted DNA are indeed undergoing apoptosis, in
contrast to the borr mutant cells that are well-integrated into the
epithelium and display only background levels of active caspase 3 staining. Cells
with low caspase staining can also be observed: these
show apparently fragmented but not yet compacted DNA, and may thus represent
an intermediate stage (Hanson, 2005).

These results, together with observations in Borr-depleted embryos and
tissue culture cells, suggest that borr mutant cells can undergo
several consecutive abnormal mitoses, which results in large polyploid cells
that eventually undergo apoptosis. Apoptotic cells appear to be cleared by
basal extrusion from the epithelium (Hanson, 2005).

Early borr mutant clones have non-autonomous effects on tissue architecture

To assess the consequences of Borr loss on the development of the imaginal
discs, borr mutant clones were induced in first or early second instar
larvae, and the resulting adult flies were examined. The most common defects in
these flies are abnormal legs and rough eyes. In addition, they often show other striking defects in tissue architecture, e.g. large wing nicks. In all these cases, a twin spot is apparent, but no mutant tissue is detectable. This indicates that, by the adult stage,
each of these early-induced borr mutant cells has undergone
apoptosis. The nature and extent of the adult defects also suggests that they
may be due partly to non-autonomous effects of the borr mutant clones
on their neighbouring wild-type tissue (Hanson, 2005).

To gain more direct evidence for these putative non-autonomous effects, the expression of Wingless (Wg) was examined in wing discs bearing borr
mutant clones: Wg is a secreted morphogen that is expressed in a thin stripe along
the developing margin of the wild-type disc and controls its formation. As
expected from the adult phenotypes, Wg expression is perturbed in various ways
by borr mutant clones. Some of the surviving giant borr mutant cells within the Wg-expressing territory cause a significant lateral expansion of Wg staining by virtue of their sheer size. Other cases of expanded Wg staining are not detectably associated with mutant cells, and thus appear to be cell non-autonomous consequences of borr loss (Hanson, 2005).

Clear non-autonomous effects of borr mutant cells
were observed if the expression of cut and senseless, two of
the ultimate target genes responding to the Wg morphogen in the marginal
region, were examined. For example, a single surviving giant borr mutant
cell expressing high levels of Cut can cause suppression of Cut and Senseless
expression in neighbouring wild-type cells. A similarly
striking example is the introduction of a V shape into the patterns of Cut and
Senseless expression caused by a borr mutant clone. The presence of a
large twin spot associated with this abnormality indicates that the causative
borr mutant clone arose early when the disc contained only a small
number of cells. Again, the borr mutant cells have disappeared in
this case, most likely through apoptosis. The kink introduced into
the expression domains of both proteins appears to coincide with a
rearrangement of cells in this region. Indeed, it appears that a single giant borr mutant cell, in the process of
basal displacement, might drag along normal epithelial cells. Thus, apoptosis
and basal extrusion of a giant cell may exert sufficient disruption of the
epithelium to induce compensatory cell rearrangements aimed at repairing
epithelial integrity, which in the event compromise patterning (Hanson, 2005).

Late borr mutant clones affect external sensory organ development

If borr mutant clones are induced late (from the early third
larval instar onwards), the resulting flies are viable and display no gross
patterning defects. Indeed, analysis of marked clones and twin spots in adult
wings suggests that all borr mutant clones are fully viable, given
that they occupy roughly the same amount of territory as their twin spots. This is somewhat unexpected in the light of results with earlier-induced clones whose
survival is severely compromised owing to abnormal mitoses. Indeed, the size of
the late-induced borr mutant clone indicates that the
mutant cells have survived three or four consecutive (abnormal) mitoses
without entering the apoptotic pathway (Hanson, 2005).

Closer examination of the flies bearing late-induced borr mutant
clones revealed that their wing blades contain clusters of hairs (trichomes)
surrounded by large clearings, rather than the usual regularly spaced single
hairs. The number of
hairs per cluster varies, with the largest cluster observed consisting of 12
hairs. All these hair clusters are produced by borr mutant cells (as
judged by their trichome marker), so this phenotype is strictly
cell-autonomous. The borr mutant clones do not significantly affect
the planar polarity in the wing blade; mutant and surrounding wild-type
hairs appear normally oriented (Hanson, 2005).

Examination of borr mutant clones in pupal wing discs supports the
notion that all late-induced borr mutant clones occupy roughly the
same amount of territory as their twin spots, confirming that the mutant cells
are fully viable at this stage. In support of this, no nuclei were observed with
compacted DNA (that would indicate imminent apoptosis). As in the larval
discs, the surviving borr mutant cells in the pupal discs are much
larger than their neighbours, often with giant nuclei, indicating a
high degree of ploidy. These giant borr mutant cells appear healthy
and are well integrated within the epithelial tissue. Their large size
provides an explanation for the observed adult phenotype, and are consistent
with a single borr mutant cell producing multiple hairs: other
conditions that produce large cells  for example, cdc2, UltA
or UltB mutant clones, or wounding  result in similar
cell-autonomous clusters of trichomes, albeit in some cases with fewer hairs
per cluster (Hanson, 2005).

Abnormal giant bristles were observed in the wing margins of flies
bearing late-induced borr mutant clones; these giant bristles
invariably lack sockets. Since it was not possible to determine whether these abnormal bristles are derived from mutant cells (owing to the weak phenotype of their bristle marker), incipient bristles were visualized in the pupal wing by ß-tubulin antibody staining. This revealed large borr mutant trichogen cells (identifiable by their lack of GFP) that generate bristles twice the normal
size. In addition,
unlike wild-type bristles, these giant bristles do not exhibit any
ß-tubulin accumulation at their bases, confirming that the
developing socket is absent around the borr mutant bristles (Hanson, 2005).

Bristles are part of sensory organs, which are composed of four cells
-- the trichogen (bristle-producing cell), tormogen (socket-producing
cell), neuron and thecogen (sheath cell); these are the progeny of a single
sensory organ precursor cell produced by consecutive invariant lineage
divisions. Evidently, loss of borr compromises the
lineage-generating divisions, and the single polyploid mutant cell seems to
develop invariably as a trichogen at the expense of the tormogen and,
possibly, of the other two sensory organ cells (Hanson, 2005).